1. Field of the Invention
The present invention relates to methods for detection of prion protein (also denoted as PrP-Sc protein) as an indicator of transmissible spongiform encephalopathies. In particular, this invention relates to non-invasive, preclinical methods for detection of prion proteins in ruminants using third eyelid lymphoid tissue. The invention further relates to monoclonal antibodies that specifically bind a conserved epitope of prion proteins in ruminants and immunoassays using the antibodies to detect prion protein in fixed, treated tissue.
2. Description of the Art
Transmissible spongiform encephalopathies (TSEs) are a heterogeneous group of fatal neurodegenerative disorders that occur in humans, ruminant herbivores, mink, and cats. Sheep scrapie is the prototype of this group. TSEs are characterized by deposition of prion proteins (also denoted as PrP-Scrapie or PrP-Sc), the infectious form of the proteins, in the central nervous system of affected individuals. Prions have been defined as small proteinaceous infectious particles which resist inactivation by procedures that modify nucleic acids. The term xe2x80x9cprionxe2x80x9d is a contraction of the words xe2x80x9cproteinxe2x80x9d and xe2x80x9cinfection,xe2x80x9d and prions are comprised largely if not exclusively of PrP-Sc molecules encoded by a PrP gene. Prion diseases are often called spongiform encephalopathies because of the post mortem microscopic or histopathologic appearance of the brain of an infected animal with large vacuoles in the cortex and cerebellum. Prion proteins are insoluble, protease-resistant glycoproteins resulting from post translational modification of normal mammalian glycoproteins (PrP-Cellular or PrP-C), and deposition of the prion protein, an abnormal isoform of a native cellular sialoglycoprotein, in the central nervous system is a reliable marker of TSE infection.
The most widely studied TSEs in food-producing animals include scrapie in sheep and goats, bovine spongiform encephalopathy (BSE) in cattle (also known as xe2x80x9cMad Cowxe2x80x9d disease), and chronic wasting disease (CWD) in mule deer and elk. Other TSEs in animals included transmissible mink encephalopathy (ILE) in mink and feline spongiform encephalopathy (FSE) of cats. Prion diseases of humans have also been identified. These include: Creutzfeldt-Jakob Disease (CJD); Gerstmann-Straussler-Scheinker Syndrome (GSS); Fatal Familial Insomnia (FFI), and Kuru.
The transmissible agent in these diseases remains controversial. However, as noted above, an insoluble isoform, (prion or PrP-Sc) of a mammalian sialoglycoprotein (Prp-Cellular or PrP-C) is a major component in infectious material. It appears that the scrapie isoform of the prion protein (PrP-Sc) is necessary for both the transmission and pathogenesis of the transmissible neurodegenerative diseases of animals and humans (see S. B. Prusiner, Science 252:1515-1522 (1991)). A leading hypothesis is that prion diseases result from the conversion of PrP-C to Prp-Sc by a nucleation or polymerization event.
The occurrence of novel transmissible spongiform encephalopathies in cattle in the United Kingdom and Europe and in mule deer and elk in parts of the United States has emphasized the need for reliable diagnostic tests. Further, the epizootic of a TSE in cattle and its postulated relationship to a new variant of human Creutzfeldt Jakob Disease have increased public and scientific awareness of these relatively rare disorders, and have highlighted the need for preclinical detection of TSEs. Although no cases of BSE have been detected in the United States, sensitive immunohistochemical techniques and preclinical detection methods are basic for detection, surveillance, and control of TSEs.
Prion diseases can have a long incubation period. For example, in sheep it can take 3 to 5 years from the time when an animal becomes infected until it first shows disease signs. In bovine spongiform encephalopathy (BSE) it can take two to eight years from the time when an animal becomes infected until it first shows disease signs. Infected animals and humans have neither a disease-specific immune response nor consistent biochemical, hematological and gross pathological abnormalities. The early diagnosis of transmissible spongiform encephalopathies can therefore be dependent on the appearance of clinical signs, electroencephalography, or the invasive method of taking brain biopsies. Confirmation of TSEs is accomplished by postmortem microscopic or histological examination of brain tissue of suspected cases. Postmortem histopathologic diagnosis of the ruminant TSEs is based on the appearance of neuronal vacuolation, spongiform changes, gliosis, and astrocytosis. However, these can vary in intensity and anatomic location depending on the host species, the individuals, host genetics, stage of disease, and infectious source. Thus, diagnosis by histopathology alone may be equivocal in early cases and usually not possible in autolyzed tissue.
Deposition of prion protein (PrP-Sc) in the central nervous system is a reliable marker for the TSEs. Immunohistochemical detection of PrP-Sc is therefore an important adjunct to histopathology in diagnosis, surveillance, and control of TSEs. Monoclonal antibody 263K 3F4 (U.S. Pat. No. 4,806,627) detects PrP-Sc in hamsters and humans, and has received widespread use in diagnostic assays and pathogenesis studies of human TSEs. A major disadvantage is that it fails to react with PrP from sheep and cattle (R. J. Kascsak et al., Immunological Investigations 26:259-268 (1997)). Rabbit antisera reactive with ruminant Prp-Sc has the disadvantages that it cannot be standardized for widespread use due to limitations in quantity and specificity. M. Horiuchi et al. (Journal of General Virology 76:2583-2587 (1995)) describe a panel of synthetic peptides that generated monoclonal and polyclonal antibodies reactive with the PrP-Cellular, the non-disease-related protein) in immunoblots of selected sheep and cattle tissue. They did not report effectiveness for detecting the disease-related isoform, PrP-Sc. Additionally, they did not they report effectiveness in detecting either PrP-C or PrP-Sc in formalin fixed tissues.
Post mortem diagnosis of prion diseases is made using histologic and immunohistochemical assays on brain tissue. Ante-mortem testing in humans with suspected CJD is performed by immunohistochemical and histologic examination of brain biopsies. Because brain biopsy in ruminant animals is not feasible, an alternative approach, based on W. J. Hadlow et al.""s observation (The Journal of Infectious Diseases 146:657-664 (1982)), has been to biopsy selected lymph nodes. Hadlow et al. demonstrated that infectivity was detectable in certain lymph nodes (retropharyngeal, tonsil, mesenteric, prescapular, bronchial-mediastinal, and spleen) and the lymphoid tissue in the intestine of scrapie-infected sheep. Hadlow""s studies, carried out before the discovery of the prion protein, detected infectivity by mouse inoculation. Race et al. (American Journal of Veterinary Research 53:883-889 (1992)), Ikegami et al. (Veterinary Record 128:271-275 (1991)), and van Keulen et al. (Journal of Clinical Microbiology 34:1228-1231 (1996)) performed similar surveys by Western immunoblots or immunohistochemical assay of selected lymph nodes using polyclonal antisera. Major disadvantages of these procedures include the following: sampling of these internal tissues requires expensive invasive methods including general anesthesia with its concomitant risks and recovery period; lymphoid tissues of sheep are often infected with a bacteria, Corynebacterium pseudotuberculosis, which destroys the architecture of the node and limits its use in these assays; and tonsillar tissue traps environmental antigens, including fungal antigens, some of the which cross react with PrP-Sc, giving equivocal or false positive immunohistochemical reactions which must be resolved by technically demanding Western blot analysis.
The BSE epidemic in the United Kingdom and the European community has cost producers and consumers in direct livestock losses and indirect loss of markets for beef and beef by-products, including economically important pharmaceutical products. Sheep and beef producing countries around the world are conducting costly surveillance and quarantine programs to maintain their status as BSE-free. Most importantly, data from several scientific lines of inquiry have provided strong evidence that BSE has infected humans in Great Britain. The scope of this new disease has yet to be determined.
What are needed is a practical, inexpensive, non-invasive method for detection of PrP-Sc in live animals and sensitive immunohistochemical assays to detect PrP-Sc in animal tissues and animal by-products.
The present invention relates to methods for detection of prion or PrP-Sc proteins as an indication of transmissible spongiform encephalopathies. In one embodiment, the invention comprises a non-invasive diagnostic assay using third eyelid lymphoid tissue to detect PrP-Sc in ruminants. This method is the first report of the use of third eyelid lymphoid tissue to detect any infectious organism. Further, this is the first report of PrP-Sc in the third eyelid lymphoid tissue.
The third eyelid represents an easily obtainable specimen for testing tissue from live animals or from animals sampled at slaughter. Thus, this detection method provides a much needed practical method for early detection of PrP-Sc and provides a means for preclinical diagnosis of TSEs.
In a second embodiment, the invention comprises monoclonal antibodies that specifically bind a conserved epitope on the ruminant prion proteins. The monoclonal antibodies of the invention react with prion protein in tissues from sheep, cattle, mule deer, and elk with naturally occurring TSE. Additionally, the antibodies detect PrP-Sc in fixed, treated tissue as an indication of the presence of TSE infection, and provide a sensitive reagent for diagnosis of TSEs.
The invention further includes immunoassay methods using the antibodies, including immunohistochemistry assays, Western immunoblots, and dot blots.
In accordance with this discovery, it is an object of the invention to provide methods for detection of prion or PrP-Sc as a marker for TSEs, including preclinical detection of infected live animals, and postmortem detection methods.
Another object of the invention is the provision of a non-invasive diagnostic assay based on biopsy of third eyelid lymphoid tissue and detection of PrP-Sc in situ as a practical method for early detection of PrP-Sc.
A further object of the invention is to provide monoclonal antibodies which recognize a conserved epitope in formalin fixed paraffin sections after treatment, including hydrated autoclaving. These monoclonal antibody reagents to conserved epitopes on PrP-Sc provide specific, reliable, and flexible tools for the accurate diagnosis of TSE. Uses of the antibodies include as reagents for standardized diagnostic testing and comparative pathology studies.
A still further object comprises immunoassay methods useful in diagnostic and pathogenesis studies of TSE in ruminants, and useful for detection, surveillance, and control of TSEs.
Other objects and advantages of the invention will become readily apparent from the ensuing description.
In one embodiment, the invention comprises a non-invasive diagnostic assay using third eyelid lymphoid tissue to detect PrP-Sc in ruminants. The nictitating membrane or third eyelid (palpebra tertia) of ruminant animals consists of a cartilaginous sheet with superficial lymphoid follicles and a seromucinous secretory gland beneath the conjunctiva of the bulbar surface. Ruminant animals including sheep, goats, mule deer, elk, and cattle have third eyelids.
In the practice of the invention, a sample of nictitating membrane-associated lymphoid tissue is collected from the animal to be tested. This can be readily carried out by everting the third eyelid. Typically two clusters of lymphoid tissue are visualized superior to the more pale glandular tissue. Biopsy of the lymphoid nodule can be performed using only local anesthetic.
The collected tissue sample is then subjected to immunohistochemical or other protein-detecting methods which are capable of detecting prion or PrP-Sc, if present in the tissue. For purposes of this invention, the term prion or PrP-Sc is defined as the disease-related protein that is a marker of TSEs.
Detection methods include immunoassays using polyclonal or monoclonal antibodies that specifically bind an epitope of PrP-Sc, for example, immunohistochemistry assays, Western immunoblots, and dot blots.
A preferred detection method comprises immunoassays using a monoclonal antibody that specifically binds a conserved epitope on the ruminant prion proteins. This antibody is described in detail below in the second embodiment of this invention.
Also preferred is the sensitive immunohistochemical technique described in detail, below, in Example 1. In brief, a fixed tissue section is treated to unmask the epitope to PrP-Sc and enhance antibody binding, and to eliminate availability of the corresponding epitope of PrP-Cellular which is expressed in tissues from normal animals. This can be conveniently carried out by (a) hydrated autoclaving, e.g., by autoclaving hydrated sections in water or buffer at about 121xc2x0 C. for 20 to 30 minutes, followed by cooling; (b) treatment with 98% formic acid for 30 minutes with or without a subsequent step of hydrated autoclaving, or (c) digestion with trypsin (e.g., 0.1% trypsin for 20 minutes at 37xc2x0 C. in Tris HCl buffer, pH 7.6). The sample is then incubated with antibody that specifically binds PrP-Sc for a time and under conditions effective to bind PrP-Sc, if present in the tissue.
Bound antibody is detected using known methods. In the preferred embodiment, detection is carried out by two cycles of incubation with a second antibody, e.g., biotinylated anti-mouse IgG, and labeled biotin, e.g., avidin-biotin-horseradish peroxidase complex, with intervening washes in buffer, e.g., Tris-HCl. An indicator such as a chromogen is added to detect bound antibody.
As shown in Example 1, below, using nictitating membrane-associated lymphoid tissue, PrP-Sc was detected in clinically ill (scrapie-infected) and in clinically normal high risk sheep aged 1, 2, and 3 years.
In a second embodiment, the invention comprises monoclonal antibodies that specifically bind a conserved epitope on the ruminant PrP proteins in fixed or frozen tissue that has been treated to unmask the epitope to PrP-Sc and eliminate availability of the corresponding epitope of PrP-C. The monoclonal antibodies of the invention react with PrP-Sc protein in tissues from sheep, cattle, mule deer, and elk with naturally occurring TSE. Presence of PrP-Sc indicates the scrapie-, bovine encephalopathy- or chronic wasting disease-infected animals.
Monoclonal antibodies encompassed by this invention bind the conserved epitope of the PrP gene product in ruminants identified as Ile-His-Phe-Gly. This epitope is further identified as comprising amino acids 142-145 of the ovine (sheep) PrP gene product, which are identical to amino acids 142-145 of the cervid (mule deer and Rocky Mountain elk) PrP gene product and amino acids 150-153 of the bovine PrP gene product. A critical feature of the antibodies of the invention is that they have the further property that they detect the unmasked epitope to PrP-Sc protein in fixed or frozen tissue. Thus, the monoclonal antibodies of the invention provide a sensitive reagent for diagnosis of TSEs.
Exemplary of the monoclonal antibodies of the invention that binds the conserved epitope Ile-His-Phe-Gly of PrP-Sc in fixed or frozen, treated ruminant tissue is monoclonal antibody F89/160.1.5. The isotype of this antibody is IgGl. The immunohistochemical staining pattern of monoclonal antibody F89/160. 1.5 was similar to patterns described in the brain of scrapie-affected sheep using polyclonal rabbit antisera to ovine or hamster PrP.
Antibodies of the invention were obtained as described in Example 2, below. In brief, we synthesized a peptide representing bovine residues 146-159, and coupled it to maleimide-activated KLH for use as an immunogen. Antisera and monoclonal antibodies from inoculated mice were screened for reactivity to recombinant sheep PrP to select cross-reacting antibodies. These antibodies were then shown to be reactive with PrP-Sc in formalin fixed, hydrated autoclaved tissue of sheep, cattle, mule deer and elk.
For purposes of this invention, monoclonal antibodies that specifically bind the conserved epitope of PrP-Sc encompassed by this invention are those which are specific for the conserved epitopes of the ruminant PrP-Sc protein, comprising Ile-His-Phe-Gly, as described above, and detect PrP-Sc in fixed or frozen, treated tissue.
The continuous cell line (hybridoma) which produces and secretes monoclonal antibody F89/160.1.5 was deposited on Sep. 24, 1997, with the American Type Culture Collection (ATCC), 1801 University Boulevard, Manassas, Va. 20110-2209 USA, under terms of the Budapest Treaty, and has been assigned accession number ATCC HB-12403.
Another exemplary monoclonal antibody prepared as described in Example 2, below, and which specifically binds the epitope to which monoclonal antibody F89/160.1.5 is directed is monoclonal antibody F89/193.1.5. The continuous cell line (hybridoma) which produces and secretes monoclonal antibody F89/1.93.1.5 was deposited May 25, 1999, with the American Type Culture Collection (ATCC), 1801 University Boulevard, Manassas, Va. 20110-2209 USA, under terms of the Budapest Treaty, and has been assigned accession number ATCC PTA-114.
The antibodies detect PrP-Sc in fixed or frozen, treated tissue as an indication of the presence of TSE infection, and provide a sensitive reagent for diagnosis of TSEs. Tissue samples useful for testing for PrP-Sc include third eyelid-associated lymphoid tissue, brain necropsy tissue, lymph node biopsy or necropsy tissue or spleen biopsy or necropsy tissue. The monoclonal antibody forms an antigen-antibody complex and the bound antibody is detected by immunological means such as enzyme-linked immunoabsorbent assay, Western blot assay, dot blot assay, immunodecoration and immunocytochemistry.
Immunoassay methods using the antibodies are also encompassed by the invention. In brief, to detect PrP-Sc, a tissue sample is obtained from an animal to be tested; the tissue is fixed, for example, by preserving in formalin or paraformaldehyde as known in the art. Next, the fixed tissue section is treated to unmask the epitope to PrP-Sc and eliminate availability of the corresponding epitope of PrP-Cellular which is expressed in tissues from normal animals, as discussed in detail above. The fixed, treated tissue is contacted with the monoclonal antibody of the invention in an amount and under conditions effective to bind PrP-Sc protein if present in the tissue. As noted in the examples, incubation of a tissue sample with about 3 xcexcg/ml of the monoclonal antibody in buffer overnight at room temperature causes antibody to be bound to the conserved PrP-Sc epitope. The antibody is detected by procedures known in the art as discussed above. In one aspect, the monoclonal antibody bound to the tissue sections is detected by contacting it with a detectably labeled second antibody under conditions such that the second antibody binds to the monoclonal antibody and detecting the second antibody so bound. In a still further aspect, the monoclonal antibody bound to the tissue sections is detected by contacting it with a labeled second antibody under conditions such that the second antibody binds to the monoclonal antibody; then the second antibody is contacted with an enzyme-ligand complex such that the complex binds to the label on the second antibody, and the enzyme-ligand complex is detected, for example, with a chromogenic substrate.
Alternatively, frozen tissue is homogenized in detergent and treated with proteinase K to eliminate the 35K PrP-C band and reveal the characteristic multiple 28-32K bands of proteinase K-resistant PrP-Sc fragments. Treated proteins are separated on polyacrylamide gels and transferred to filters. The filter is contacted with the monoclonal antibody of the invention in an amount and under conditions effective to bind PrP-Sc protein if present in the tissue. The antibody is detected as described above except that the preferred final detection step is with a cherniluminescent substrate.
As discussed above, antibodies of the invention are useful in diagnostic and pathogenesis studies of the TSEs.